Mutating two putative phosphorylation sites on ZHP-3 does not affect its localization or function during meiotic chromosome segregation

Meiotic chromosome segregation depends on crossover recombination to link homologous chromosomes together and promote accurate segregation in the first meiotic division. In Caenorhabditis elegans, a conserved RING finger protein, ZHP-3, is essential for meiotic recombination and localizes to sites of crossover formation. Whether ZHP-3 is regulated to promote recombination remains poorly understood. In vitro analysis identified two putative CHK-1 kinase phosphorylation sites on ZHP-3. However, mutation of the phosphorylation sites identified in vitro had no effect on meiotic recombination or localization of ZHP-3. Thus, these two phosphorylation sites appear to be dispensable for ZHP-3’s role in meiotic recombination or its localization.


Description
Haploid sex cells such as sperm and eggs depend on a specialized form of cell division called meiosis. Throughout prophase of meiosis, homologous chromosomes pair, synapse, and recombine to form a physical link between them called a chiasma. Chiasmata are essential for proper segregation (Page et al. 2003). Defects in meiotic recombination can lead to gametes that contain an incorrect number of chromosomes (aneuploidy), which can cause infertility, miscarriages, and genetic disorders such as Down Syndrome. Therefore, identifying how recombination is regulated is crucial for understanding multiple aspects of human reproductive health.
Previous research in C. elegans has shown that several proteins called crossover factors localize to the designated crossover site to promote recombination (Bhalla et al. 2008, Yokoo et al. 2012. One of these crossover factors, ZHP-3 (Zip homologous protein 3), is highly conserved, has a budding yeast counterpart that acts as a SUMO ligase (Cheng et al. 2006) and is essential for crossover formation (Jantsch et al. 2004). Therefore, when ZHP-3 is absent, chromosome pairs are not linked by chiasmata. Because sex determination in C. elegans is based on the number of X chromosomes, with hermaphrodites having two X chromosomes while males have one, a high incidence of male (HIM) phenotype is diagnostic for chromosome segregation errors. Loss of ZHP-3 results in a high rate of embryonic lethality and a HIM phenotype (Jantsch et al. 2004), both indicating chromosome segregation errors. We've shown that ZHP-3's localization is dynamic throughout prophase: in mid-pachytene ZHP-3 is found all along the synaptonemal complex (SC) between synapsed homologous chromosomes. During late pachytene, it re-localizes and concentrates at the crossover site (Bhalla et al. 2008). How ZHP-3 achieves this dynamic localization to promote meiotic recombination is currently unknown.
Meiotic proteins are phosphorylated to promote essential steps in meiotic prophase, including stabilizing the SC (Nadarajan et al. 2017), designating the short arm of chromosome pairs to promote two step cohesion loss (Sato-Carlton et al. 2018), and activating negative feedback loops in response to defects in synapsis or crossover formation (Kim et al. 2015). CHK-1 (Checkpoint Kinase 1) localizes to early and mid-pachytene nuclei (Woglar et al. 2013), correlating with when ZHP-3 is present along the SC. Similar to SC proteins that are known to be phosphorylated (Nadarajan et al. 2017, Sato-Carlton et al. 2018, ZHP-3 also contains an unstructured C-terminal tail containing multiple serine residues (Reynolds et al. 2013). Here we test whether CHK-1 is a potential regulator of ZHP-3's localization and if phosphorylation of two residues on ZHP-3 is required for its dynamic localization and/or to promote crossover formation.
To determine if ZHP-3 is post-translationally modified, we generated truncated versions of ZHP-3 protein, purified these from BL21 codon plus cells, and performed in vitro kinase assays with radiolabeled P 32 and human Chk1 to determine if ZHP-3 is phosphorylated. We found that when ZHP-3 samples were incubated with human Chk1 and P 32 , ZHP-3 is robustly phosphorylated on its C-terminus but not its N-terminus ( Figure 1A). To identify the phosphorylated residues on ZHP-3, we sent samples for mass spectrometry and identified serine 308 and serine 366 as the phosphorylated residues ( Figure 1A).
To determine the significance of these phosphorylation events on ZHP-3's function and meiotic recombination in vivo, we used CRISPR/Cas9 genome editing (Arribere et al. 2014, Paix et al. 2015 to mutate serines 308 and 366 to alanines (zhp-3 S308A and zhp-3 S366A ) so that they can no longer undergo phosphorylation. Along with generating the single phosphorylation mutant strains, we also created the double phosphorylation mutant (zhp-3 S308A, S366A ) to test for redundancy between these two sites.
To determine if these mutations result in defects in ZHP-3's localization, we first stained mid-pachytene nuclei with antibodies against ZHP-3 and SYP-1 to determine if ZHP-3 phosphorylation mutants can still localize along the SC. In wildtype germlines, ZHP-3 is found all along the SC and colocalizes with SYP-1 (Bhalla et al. 2008, Figure 1B). We found that ZHP-3 S308A , ZHP-3 S366A and ZHP-3 S308A, S366A colocalized with SYP-1 on the SC ( Figure 1B), similar to wildtype ZHP-3, indicating that these mutations do not affect ZHP-3's ability to localize to the SC.
We also stained germlines with antibodies against HTP-3 and HTP-1, meiotic chromosomal proteins that preferentially localize to the long and short arms of bivalents (Martinez-Perez et al. 2008) to determine if there were defects in bivalent morphology. HTP-3 localizes to both the long and short arm of bivalents in a cruciform pattern, and HTP-1 localizes to the long arm on wildtype bivalents ( Figure 1E, Martinez-Perez et al. 2008). In contrast, HTP-3 and HTP-1 are lost on the long arm in zhp-3(jf61) null mutants ( Figure  1E). We found that HTP-3 localizes to both the long and short arm and HTP-1 localizes to the long arm properly in zhp-3 S308A , zhp-3 S366A , and zhp-3 S308A, S366A mutants ( Figure 1E), indicating that bivalent morphology is unaffected in zhp-3 phosphorylation mutants.
Finally, we tested whether mutation of these sites results in chromosome missegregation by assessing the number of viable and male progeny for each phosphorylation mutant strain. Males are found in ~1/1000 progeny in wildtype strains, while zhp-3(jf61) null mutants have reported 1.3% viable progeny and ~30% males (Bhalla et al. 2008). We found that both the single phosphorylation mutants and double phosphorylation mutants had 100% viable progeny and 0.03%, 0.2%, and 0.1% male progeny respectively ( Figure 1F), illustrating that mutation of these sites does not cause general defects in meiosis.
Regulation of meiotic recombination is essential for proper development of gametes and preventing aneuploidy. While ZHP-3 is essential for meiotic recombination in C. elegans, whether and how it is regulated to promote recombination remains an active area of investigation. While we found that ZHP-3 is phosphorylated on its C-terminus at S308 and S366 in vitro, mutation of these phosphorylation sites in vivo does not result in defects in ZHP-3's localization ( Figures 1B and 1C), bivalent formation ( Figures 1D  and 1E), or chromosome segregation ( Figure 1F).
We conclude that ZHP-3 is either not regulated by these phosphorylation sites or that there is potential redundancy for ZHP-3 regulation by phosphorylation. Whether ZHP-3 is modified at other residues by phosphorylation or other post-translational modifications remain open questions in the field. It has also been recently discovered that there are additional ZHP proteins that play a role in crossover formation in C. elegans (Nguyen et al. 2018, Zhang et al. 2018. How these proteins coordinate to promote recombination and whether they require protein modifications are interesting questions for determining how crossover formation is regulated to promote meiotic chromosome segregation.

C. elegans strains and genetics:
The Bristol N2 C. elegans strain (Brenner et al. 1974) was used as the wildtype control for all experiments and as the parent strain for the single phosphorylation mutant CRISPR/Cas9 genome editing experiments. The zhp-3 S366A strain was used as the background to create the zhp-3 S308A ,S366A double phosphorylation mutant.
Strains were maintained at 20°C under standard conditions for all immunostaining experiments and maintained at 15°C for all CRISPR/Cas9 genome editing experiments.

In vitro Kinase Assays
Kinase assays were performed at 30ºC in 20uL reactions with 1X kinase buffer (50mM Tris pH7.4, 1mM DTT, 25mM betaglycerophosphate, 5mM MgCl2 ,10mM ATP) for 30 minutes. Reactions included 0.5uL 32 P-γ-ATP (Perkin Elmer), 0.5uL of activated Chk1 (Sigma), and 2ug of substrate. Reactions were quenched by adding 20uL 2X sample buffer and boiling for 5 minutes. Reactions were run out in duplicate on two 10% SDS-PAGE gels, one was stained with Coomassie to verify equivalent amounts of substrate were utilized, and one was dried and then exposed to a phosphor screen (GE Life Sciences) for analysis of phosphorylation.

Mass Spectrometric Analysis
In vitro assays were performed as above without radiolabeled ATP. Samples were frozen in liquid nitrogen and sent to the QB3/Chemistry Mass Spectrometry Facility at University of California, Berkeley, which performed the desalting and trypsin digest prior to mass spectrometry. Mass spectrometry produced multiple peptides of the protein sequence of the C-terminus and GST tag, indicating good coverage, and identified S308 and S366 as the in vitro phosphorylated residues.
N2 young adults were injected with a mix that included a zhp-3 specific crRNA (IDT-Alt-R TM , 100μM stock), the dpy-10 coconversion crRNA (IDT-Alt-R TM , 100μM stock) , tracrRNA (IDT-Alt-R TM , 100μM stock) a zhp-3 specific repair oligo (IDT, 50μM stock) the dpy-10 coconversion repair oligo (IDT, 50μM stock), and purified Cas9 protein (40μM stock). F1 roller and dpy progeny from injected P0's were isolated to individual plates, and non-rolling non dpy siblings from jackpot plates (> 10 transformed worms) were grouped 3-5 F1's per plate. After F2 progeny were generated, F1's were screened for the respective phosphorylation mutant allele via PCR and restriction digest. F2's from a F1 plate that had the confirmed mutant allele were singled to identify homozygotes for each phosphorylation mutation. All phosphorylation mutant strains were confirmed via sequencing. Phosphorylation mutant strains were outcrossed to N2 worms 1-2 times prior to analysis.
For creating the zhp-3 S308A, S366A strain, the same S308A sgRNA and oligo repair template above were used to create the S308A mutation in the zhp-3 S366A mutant background.

DAPI staining and Immunostaining:
Adult hermaphrodites were fixed and stained 24-26 hours post L4 larval stage as in . For visualizing bivalents in mature oocytes for quantification, adult hermaphrodites were fixed and stained 48 hours post L4 stage.
Images of immunostaining experiments were obtained using a DeltaVision Personal DV system (Applied Precision) equipped with a 100× N.A. 1.40 oil-immersion objective (Olympus), resulting in an effective XY pixel spacing of 0.064 or 0.040 μm. Z-stacks were collected at 0.2-μm Z-spacing and processed by constrained, iterative deconvolution. Imaging, image scaling, and analysis were performed using functions in the softWoRx software package. Projections were calculated using a maximum intensity algorithm. Composite images from immunostaining experiments were processed and some false coloring was performed using Fiji.

Quantification of bivalents:
Bivalent quantification was performed on worms 48 hours post L4 larval stage. Each genotype included at least 3 germlines for quantification.

Viability assay and male counts:
N2, zhp-3(jf61), zhp-3 S308A , zhp-3 S366A , and zhp-3 S308A, S366A late L4 stage worms were isolated to plates and maintained at 20 degrees. F1 embryos and oocytes were scored at 24, 48 and 72 hours post L4 larval stage. Once F1's reached the late L4 stage, viable and male progeny were scored. The total brood size, percent viability and percent HIM phenotype were calculated for each strain. A two-sided Wilcoxon-Mann-Whitney test was performed to assess statistical significance.

Figures and statistics:
Figures were assembled using Adobe Illustrator. All histograms and statistics were performed using R (R core team) with the ggplot2 package (Wickham et al. 2016). For bivalent quantification, viability assay and male counts, a two-sided Wilcoxon-Mann-Whitney test was used to determine statistical significance.